Chromosome dynamics and the proteins that channel DNA movement in vivo are critical determinants of cell replication, gene expression, genetic recombination, and Darwinian evolution. Recent studies have demonstrated that bacterial chromosomes are organized into about 400 independent domains that limit supercoiling diffusion. The primary focus of our research over the next four years will be to identify the critical proteins and dissect the mechanical mechanisms that control bacterial chromosome structure and supercoil movement inside living cells. We have specific aims. 1) Connect the activities of gyrase and the bacterial condensin, MukB, to nucleoid compaction. This aim includes a new component involving DNA gyrase biochemistry and genetic methods that evolved from the E. coli/Salmonella species comparison. Genetic selections will be used to identify the proteins that form both the stochastic and sequence-specific domain boundaries in the 400 domain chromosome. Candidate "Domainins" or proteins that control a segment of chromosome structure will be run through a gauntlet of 4 tests to measure domain behavior. These tests include analyzing specific genes for their ability to change supercoil density and site-specific resolution efficiency at eight different locations, testing their effect on ribosomal RNA operons, and measuring the average domain size for each gene. Connect domains and DNA movement to structure. We will test a loop model for domain structure and define the dynamic characteristics of highly transcribed ribosomal RNA operons. As cells grow rapidly in rich media, 70% of all RNA synthesis is devoted to stable RNAs (ribosomal and tRNA genes). New experiments will test whether these regions form specific transcription loops and determine where the highly transcribed genes are in the folded genome. We will also establish whether or not transcription in WT bacteria can generate "waves of positive supercoils." 2) Connect domains and movement to structure. We will test a loop model for organizing highly transcribed protein-encoding genes, ribosomal RNA operons, and tRNA operons. We will also exploit the chromosome conformation capture technology to prove our hypothesis on looping. 3) Connect the average chromosome structure to single cell behavior using fluorescent cell technology. The domain structure of Lac- and Tet-operator modules that serve as cell biological markers of chromosome behavior will be analyzed. Studies will determine how modules behave as chromosome dynamics elements in vivo when unoccupied, when decorated with different levels of fluorescent binding protein, and how inducer changes supercoil dynamics for sites with bound repressors. In the course of these experiments, we will place fluorescent protein binding modules into the E. coli and Salmonella chromosome at 20 different positions using efficient recombination methods developed in the last grant period, and determine what happens when a segment of chromosome is separated from the main body by site specific recombination. PUBLIC HEALTH RELEVANCE: This project aims to develop a molecular model of chromosome organization and measure DNA dynamics inside living cells. Many essential proteins that participate in bacterial cell division are also found in eukaryotic organisms up to humans. This work provides a rationale for developing new antibiotics to fight pathogenic microorganisms and to solve old problems about how chromosomes become disentangled during cell growth.